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Inhibition of Phosphatidylcholine-Specific Phospholipase C Interferes with Proliferation and Survival of Tumor Initiating Cells in Squamous Cell Carcinoma.

Cecchetti S, Bortolomai I, Ferri R, Mercurio L, Canevari S, Podo F, Miotti S, Iorio E - PLoS ONE (2015)

Bottom Line: Compared with A431-AD, A431-SPH cells showed about 2.8-fold lower PC-PLC protein and activity levels, but similar nuclear content.Exposure of adherent cells to the PC-PLC inhibitor D609 (48h) induced a 50% reduction of cell proliferation at doses comprised between 33 and 50 μg/ml, without inducing any relevant cytotoxic effect (cell viability 95±5%).Furthermore, D609 treatment of A431-AD and CaSki-AD cells affected the sphere-forming efficiency, which dropped in both cells, and induced down-modulation of stem-related markers mRNA levels (Oct4, Nestin, Nanog and ALDH1 in A431; Nestin and ALDH1 in CaSki cells).

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Neurosciences, Istituto Superiore di Sanità, Rome, Italy.

ABSTRACT

Purpose: The role of phosphatidylcholine-specific phospholipase C (PC-PLC), the enzyme involved in cell differentiation and proliferation, has not yet been explored in tumor initiating cells (TICs). We investigated PC-PLC expression and effects of PC-PLC inhibition in two adherent (AD) squamous carcinoma cell lines (A431 and CaSki), with different proliferative and stemness potential, and in TIC-enriched floating spheres (SPH) originated from them.

Results: Compared with immortalized non-tumoral keratinocytes (HaCaT) A431-AD cells showed 2.5-fold higher PC-PLC activity, nuclear localization of a 66-kDa PC-PLC isoform, but a similar distribution of the enzyme on plasma membrane and in cytoplasmic compartments. Compared with A431-AD, A431-SPH cells showed about 2.8-fold lower PC-PLC protein and activity levels, but similar nuclear content. Exposure of adherent cells to the PC-PLC inhibitor D609 (48h) induced a 50% reduction of cell proliferation at doses comprised between 33 and 50 μg/ml, without inducing any relevant cytotoxic effect (cell viability 95±5%). In A431-SPH and CaSki-SPH D609 induced both cytostatic and cytotoxic effects at about 20 to 30-fold lower doses (IC50 ranging between 1.2 and 1.6 μg/ml). Furthermore, D609 treatment of A431-AD and CaSki-AD cells affected the sphere-forming efficiency, which dropped in both cells, and induced down-modulation of stem-related markers mRNA levels (Oct4, Nestin, Nanog and ALDH1 in A431; Nestin and ALDH1 in CaSki cells).

Conclusions: These data suggest that the inhibition of PC-PLC activity may represent a new therapeutic approach to selectively target the most aggressive and tumor promoting sub-population of floating spheres originated from squamous cancer cells possessing different proliferative and stemness potential.

No MeSH data available.


Related in: MedlinePlus

Cytostatic and cytotoxic effects of D609 on A431-SPH cells.Cells were seeded 72 hours before adding different doses of D609 at t = 0 (● = untreated cells; □ = 1.5 μg/ml, ▲ = 3 μg/ml, ▼ = 6 μg/ml, ◇ = 12.5 μg/ml, * = 25 μg/ml and ○ = 50 μg/ml) and monitored for 24h and 48 h afterwards. Cell counting (mean ± SD, n = 3) of total present cells (A) and of live (white columns) and dead (black columns) cells (B) measured by Trypan blue exclusion test and by automated cell counter, as described in the Material and Methods section. Arrows in the panel B indicate the dose of D609 used for the Western blot analyses reported in D. C) Evaluation of the effect of PC-PLC inhibition on A431-SPH cell morphology and death. Images were taken on untreated cells or after 24h and 48h of D609 exposure at the indicated doses. Scale bars, 100 μm. Inserts show a 100x magnification. D) Representative Western blot analyses (of 3 independent experiments) of PC-PLC protein expression in total lysates of A431-SPH cells, cultured in the presence or absence of 1.5 μg/ml D609 for the indicated times; β-actin was used as a quantitative loading control. E) Sphere Formation Efficiency (SFE) of A431-AD cells untreated (white column) or treated with 50 μg/ml D609 for 24h (black column) or 48h (grey column) (mean ± SD; n = 4 at T0 and 24h; n = 7 at 48h). Statistical analyses were performed using one-way ANOVA, P = 0.002. F) Real Time PCR analyses of stemness markers OCT4, NANOG, NESTIN and ALDH1 expressed by A431-AD cell lines either untreated (white columns) or in the presence of D609 (50 μg/ml, black columns) for 48h. Statistical analyses were performed using t-test (OCT4, P = 0.003; NANOG, P = 0.026; NESTIN, P = 0.001; ALDH1, P = 0.009).
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pone.0136120.g005: Cytostatic and cytotoxic effects of D609 on A431-SPH cells.Cells were seeded 72 hours before adding different doses of D609 at t = 0 (● = untreated cells; □ = 1.5 μg/ml, ▲ = 3 μg/ml, ▼ = 6 μg/ml, ◇ = 12.5 μg/ml, * = 25 μg/ml and ○ = 50 μg/ml) and monitored for 24h and 48 h afterwards. Cell counting (mean ± SD, n = 3) of total present cells (A) and of live (white columns) and dead (black columns) cells (B) measured by Trypan blue exclusion test and by automated cell counter, as described in the Material and Methods section. Arrows in the panel B indicate the dose of D609 used for the Western blot analyses reported in D. C) Evaluation of the effect of PC-PLC inhibition on A431-SPH cell morphology and death. Images were taken on untreated cells or after 24h and 48h of D609 exposure at the indicated doses. Scale bars, 100 μm. Inserts show a 100x magnification. D) Representative Western blot analyses (of 3 independent experiments) of PC-PLC protein expression in total lysates of A431-SPH cells, cultured in the presence or absence of 1.5 μg/ml D609 for the indicated times; β-actin was used as a quantitative loading control. E) Sphere Formation Efficiency (SFE) of A431-AD cells untreated (white column) or treated with 50 μg/ml D609 for 24h (black column) or 48h (grey column) (mean ± SD; n = 4 at T0 and 24h; n = 7 at 48h). Statistical analyses were performed using one-way ANOVA, P = 0.002. F) Real Time PCR analyses of stemness markers OCT4, NANOG, NESTIN and ALDH1 expressed by A431-AD cell lines either untreated (white columns) or in the presence of D609 (50 μg/ml, black columns) for 48h. Statistical analyses were performed using t-test (OCT4, P = 0.003; NANOG, P = 0.026; NESTIN, P = 0.001; ALDH1, P = 0.009).

Mentions: To assess whether D609 has an active role on the TIC sub-population in squamous cancer, we evaluated the effects of D609 treatment on A431-SPH cells. Fig 5A shows that D609 was extremely potent in inhibiting the proliferation of A431-SPH cells; indeed at 3 μg/ml, i.e. at a dose 16-fold lower than that effective on parental A431-AD cells and HaCaT keratinocytes (for comparison see Fig 2B), D609 was already able to almost totally block the A431-SPH growth.


Inhibition of Phosphatidylcholine-Specific Phospholipase C Interferes with Proliferation and Survival of Tumor Initiating Cells in Squamous Cell Carcinoma.

Cecchetti S, Bortolomai I, Ferri R, Mercurio L, Canevari S, Podo F, Miotti S, Iorio E - PLoS ONE (2015)

Cytostatic and cytotoxic effects of D609 on A431-SPH cells.Cells were seeded 72 hours before adding different doses of D609 at t = 0 (● = untreated cells; □ = 1.5 μg/ml, ▲ = 3 μg/ml, ▼ = 6 μg/ml, ◇ = 12.5 μg/ml, * = 25 μg/ml and ○ = 50 μg/ml) and monitored for 24h and 48 h afterwards. Cell counting (mean ± SD, n = 3) of total present cells (A) and of live (white columns) and dead (black columns) cells (B) measured by Trypan blue exclusion test and by automated cell counter, as described in the Material and Methods section. Arrows in the panel B indicate the dose of D609 used for the Western blot analyses reported in D. C) Evaluation of the effect of PC-PLC inhibition on A431-SPH cell morphology and death. Images were taken on untreated cells or after 24h and 48h of D609 exposure at the indicated doses. Scale bars, 100 μm. Inserts show a 100x magnification. D) Representative Western blot analyses (of 3 independent experiments) of PC-PLC protein expression in total lysates of A431-SPH cells, cultured in the presence or absence of 1.5 μg/ml D609 for the indicated times; β-actin was used as a quantitative loading control. E) Sphere Formation Efficiency (SFE) of A431-AD cells untreated (white column) or treated with 50 μg/ml D609 for 24h (black column) or 48h (grey column) (mean ± SD; n = 4 at T0 and 24h; n = 7 at 48h). Statistical analyses were performed using one-way ANOVA, P = 0.002. F) Real Time PCR analyses of stemness markers OCT4, NANOG, NESTIN and ALDH1 expressed by A431-AD cell lines either untreated (white columns) or in the presence of D609 (50 μg/ml, black columns) for 48h. Statistical analyses were performed using t-test (OCT4, P = 0.003; NANOG, P = 0.026; NESTIN, P = 0.001; ALDH1, P = 0.009).
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Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4581859&req=5

pone.0136120.g005: Cytostatic and cytotoxic effects of D609 on A431-SPH cells.Cells were seeded 72 hours before adding different doses of D609 at t = 0 (● = untreated cells; □ = 1.5 μg/ml, ▲ = 3 μg/ml, ▼ = 6 μg/ml, ◇ = 12.5 μg/ml, * = 25 μg/ml and ○ = 50 μg/ml) and monitored for 24h and 48 h afterwards. Cell counting (mean ± SD, n = 3) of total present cells (A) and of live (white columns) and dead (black columns) cells (B) measured by Trypan blue exclusion test and by automated cell counter, as described in the Material and Methods section. Arrows in the panel B indicate the dose of D609 used for the Western blot analyses reported in D. C) Evaluation of the effect of PC-PLC inhibition on A431-SPH cell morphology and death. Images were taken on untreated cells or after 24h and 48h of D609 exposure at the indicated doses. Scale bars, 100 μm. Inserts show a 100x magnification. D) Representative Western blot analyses (of 3 independent experiments) of PC-PLC protein expression in total lysates of A431-SPH cells, cultured in the presence or absence of 1.5 μg/ml D609 for the indicated times; β-actin was used as a quantitative loading control. E) Sphere Formation Efficiency (SFE) of A431-AD cells untreated (white column) or treated with 50 μg/ml D609 for 24h (black column) or 48h (grey column) (mean ± SD; n = 4 at T0 and 24h; n = 7 at 48h). Statistical analyses were performed using one-way ANOVA, P = 0.002. F) Real Time PCR analyses of stemness markers OCT4, NANOG, NESTIN and ALDH1 expressed by A431-AD cell lines either untreated (white columns) or in the presence of D609 (50 μg/ml, black columns) for 48h. Statistical analyses were performed using t-test (OCT4, P = 0.003; NANOG, P = 0.026; NESTIN, P = 0.001; ALDH1, P = 0.009).
Mentions: To assess whether D609 has an active role on the TIC sub-population in squamous cancer, we evaluated the effects of D609 treatment on A431-SPH cells. Fig 5A shows that D609 was extremely potent in inhibiting the proliferation of A431-SPH cells; indeed at 3 μg/ml, i.e. at a dose 16-fold lower than that effective on parental A431-AD cells and HaCaT keratinocytes (for comparison see Fig 2B), D609 was already able to almost totally block the A431-SPH growth.

Bottom Line: Compared with A431-AD, A431-SPH cells showed about 2.8-fold lower PC-PLC protein and activity levels, but similar nuclear content.Exposure of adherent cells to the PC-PLC inhibitor D609 (48h) induced a 50% reduction of cell proliferation at doses comprised between 33 and 50 μg/ml, without inducing any relevant cytotoxic effect (cell viability 95±5%).Furthermore, D609 treatment of A431-AD and CaSki-AD cells affected the sphere-forming efficiency, which dropped in both cells, and induced down-modulation of stem-related markers mRNA levels (Oct4, Nestin, Nanog and ALDH1 in A431; Nestin and ALDH1 in CaSki cells).

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Neurosciences, Istituto Superiore di Sanità, Rome, Italy.

ABSTRACT

Purpose: The role of phosphatidylcholine-specific phospholipase C (PC-PLC), the enzyme involved in cell differentiation and proliferation, has not yet been explored in tumor initiating cells (TICs). We investigated PC-PLC expression and effects of PC-PLC inhibition in two adherent (AD) squamous carcinoma cell lines (A431 and CaSki), with different proliferative and stemness potential, and in TIC-enriched floating spheres (SPH) originated from them.

Results: Compared with immortalized non-tumoral keratinocytes (HaCaT) A431-AD cells showed 2.5-fold higher PC-PLC activity, nuclear localization of a 66-kDa PC-PLC isoform, but a similar distribution of the enzyme on plasma membrane and in cytoplasmic compartments. Compared with A431-AD, A431-SPH cells showed about 2.8-fold lower PC-PLC protein and activity levels, but similar nuclear content. Exposure of adherent cells to the PC-PLC inhibitor D609 (48h) induced a 50% reduction of cell proliferation at doses comprised between 33 and 50 μg/ml, without inducing any relevant cytotoxic effect (cell viability 95±5%). In A431-SPH and CaSki-SPH D609 induced both cytostatic and cytotoxic effects at about 20 to 30-fold lower doses (IC50 ranging between 1.2 and 1.6 μg/ml). Furthermore, D609 treatment of A431-AD and CaSki-AD cells affected the sphere-forming efficiency, which dropped in both cells, and induced down-modulation of stem-related markers mRNA levels (Oct4, Nestin, Nanog and ALDH1 in A431; Nestin and ALDH1 in CaSki cells).

Conclusions: These data suggest that the inhibition of PC-PLC activity may represent a new therapeutic approach to selectively target the most aggressive and tumor promoting sub-population of floating spheres originated from squamous cancer cells possessing different proliferative and stemness potential.

No MeSH data available.


Related in: MedlinePlus